High-strength biological scaffold material and preparation method thereof

ABSTRACT

A high-strength biological scaffold and preparation method thereof. The biological scaffold is comprised of a framework of boiled-off silk woven material, the form of the framework is determined by actual needs; the framework surface is coated with a layer of silk protein scaffold material, fibroin protein/gelatin biological scaffold material or fibroin protein/collagen biological scaffold material having a thickness of 100 micrometers to 5 centimeters. The high-strength biological scaffold material has high tear resistance strength and mechanical strength and good biocompatibility, has a porous structure suitable for tissue regeneration, and can be used for preparing anal fistula repair plugs.

The present application claimed priority based on the patent applicationentitled by “High-strength biological scaffold material and preparationmethod thereof” filed with the Chinese Patent Office (No.201110029805.5) on Jan. 27, 2011, all contents of which are combined inthe present application by reference.

FIELD OF INVENTION

The present invention relates to a method for preparing biologicalscaffolds, particularly relates to a high-strength biological scaffoldmaterial having a porous structure suitable for tissue regeneration,prepared using silk fibroin as a raw material. This biological scaffoldmaterial can be specifically used to prepare anal fistula repair plug.It can be applied not only to the treatment of anal fistula disease, butalso to repair of both intestinal fistula and other tissue defects.

BACKGROUND OF THE INVENTION

Anal fistula is a common anorectal disease, accounting for 1.6-3.6% ofanorectal diseases, and usually formed by rupture or incision ofanorectal abscess. Compared with other diseases, anal fistula ischaracterized in extreme pain in patients, low surgical success rate,and probable recurrence, bringing about tremendous physical andpsychological problems to the patients. Anal fistula treatment mainlycomprises surgical incision, hanging line therapy, anal fistulaexcision, cryotherapy, and electrotherapy, etc. These treatments sufferfrom the defects of extreme pain during treatment, low cure rate (lowerthan 40%), long recovery period, and frequent recurrence. In recentyears, based on findings in tissue engineering study, the use ofbiocompatible scaffolds for anal fistula repair bring a new dawn to thetreatment of anal fistula. Compared with the traditional treatmentmethods, scaffold repair surgery has the advantages of reduced pains,improved success rate, fewer complications, lower recurrence rate, andhas been generally accepted by patients in developed countries. In thecurrent clinical trials, the prosthetic materials mainly used includefibrin gels and porcine small intestinal submucosa scaffold developed byCook Medical, Inc. Among them, fibrin gels degrade rapidly, and yield ananal fistula cure rate of below 40%. Compared with fibrin gels, porcinesmall intestinal submucosa scaffold has improved degradation rate, andyields an increased anal fistula cure rate to about 50%. Nevertheless,its degradation rate still cannot fully meet the requirements of thetreatment of anal fistula, and resulted in an anal fistula recurrencerate of 10% or greater. Moreover, it suffers from complex extraction andpreparation process, high cost, and high price of around $2,000 each,which is unbearable to patients in China. Thus, the treatment of analfistula is urgent for the latest results in the tissue engineeringstudies, so as to develop new porous scaffolds having goodbiocompatibility, high mechanical strength, low degradation rate, andlow price.

Silk fibroin is the main component of silk, which is inexpensive andeasy to purify. Studies have shown that silk fibroin is non-toxic,non-immunogenic, well biocompatible, biodegradable, and excellent inmechanical properties. It is an ideal raw material for the preparationof tissue repair and tissue engineering scaffolds. Silk fibroin hasdifferent crystal structure including Silk I and Silk II. The differencein types and contents of crystals can determine the solubility anddegradation of silk fibroin. By adjusting its crystal and non-crystalstructure, the in vivo degradation rate of silk fibroin can be reducedfrom one year to about half a month, so as to meet the different needsof tissue repair and regeneration.

Currently, researchers have developed various methods for preparation ofsilk fibroin scaffolds, comprising a freeze-drying method, a phaseseparation method, a salting-out method and an electrostatic spinningmethod, etc. However, the above methods each have deficiencies difficultto overcome. For example, a silk fibroin scaffold prepared bysalting-out typically has a pore size of 400 microns or more, and aporous material prepared by electrostatic spinning typically has a poresize of 100 microns or less. As such, there are restrictions for theiruse as a tissue repair or tissue engineering scaffold. A porous scaffoldhaving a larger range of pore size can be prepared by freeze-drying.However, during the freezing process, the silk fibroin is liable to beself-assembled into a sheet structure, and it is difficult to obtain agood porous structure. In the prior art, a porous scaffold having a goodpore structure and suitable for tissue growth has been successfullyprepared by controlling the self-assembly of silk fibroin. However, thescaffold thus obtained faces a major problem in practical applications.It has relatively poor mechanical strength and weak tearing resistance,and therefore is difficult to be sutured and fixed by a surgical threador to be operated by minimally invasive surgical operation, whichresults in that the scaffold cannot be practically used in clinicalapplication, especially in anal fistula repair plugs.

Therefore, there is a need to overcome the above problems in the priorart, to develop a high-strength biological scaffold material having hightear resistance strength and suitable to be sutured and fixed by asurgical thread, and thereby to prepare a fibroin anal fistula repairplug in order to meet the practical needs.

SUMMARY OF THE INVENTION

The present invention is aimed at providing a high strength biologicalscaffold material and method for preparation thereof, by improving themechanical strength and tear resistance of silk fibroin-based scaffoldin order to meet the practical needs.

In order to achieve the above purpose, the invention provides thefollowing technical solution: a high-strength biological scaffoldmaterial, characterized in that, said high-strength biological scaffoldmaterial is comprised of a coating layer and a framework, said frameworkis embedded in the coating layer; said framework is a boiled-off silkwoven material, the weave density can be determined by needs; thecoating layer has a thickness of 100 microns to 5 cm, and the materialof the coating layer is selected from the group consisting of: silkprotein scaffold material, silk fibroin/gelatin biological scaffoldmaterial or silk fibroin/collagen biological scaffold material.

In the above embodiment, the weave density of the boiled-off silk wovenmaterial is such that the distance between adjacent boiled-off silks isbetween 0.5 mm and 3 mm. For example, the distance between two adjacentwarp boiled-off silks is 0.5 mm to 3 mm, and the distance between twoadjacent weft boiled-off silks is 0.5 mm to 3 mm. A suitable weavedensity can ensure both high tear resistance of the material andsuitable porosity, and is beneficial to tissue repair and regeneration.

In the above embodiment, preferably, the coating layer has a thicknessof 100 microns to 5 mm; and the high-strength biological scaffoldmaterial has a pore size of 200 to 400 microns, and a porosity of 80% ormore.

A method for preparing the above high-strength biological scaffoldmaterial comprises the following steps: boiled-off silk obtained bydegumming silk is woven into a woven material by a textile machine toform a framework according to the desired shape, and then the frameworkis placed into a mold; a solution containing silk fibroin is injectedinto the mold having the framework placed therein, and subjected tofreezing and vacuum treatment; thereby a coating layer having athickness of 100 microns to 5 cm is formed on the surface of theframework, and the high-strength biological scaffold material isprepared, wherein said solution containing silk fibroin is one selectedfrom the group consisting of: an aqueous silk fibroin solution, a mixedsolution of silk fibroin and gelatin, and a mixed solution of silkfibroin and collagen; the weave density of the woven material is suchthat the distance between adjacent boiled-off silks is between 0.5 mmand 3 mm, and the material of the coating layer is selected from thegroup consisting of: silk protein scaffold material, silkfibroin/gelatin biological scaffold material or silk fibroin/collagenbiological scaffold material.

In the above embodiment, the step of coating the surface of theframework with a coating layer comprises the specific steps of:

1) preparing a solution containing silk fibroin, the solution containingsilk fibroin being one selected from the group consisting of: an aqueoussilk fibroin solution, a mixed solution of silk fibroin and gelatin, anda mixed solution of silk fibroin and collagen; injecting the solutioncontaining silk fibroin into the mold having the framework placedtherein, and subjecting it to freezing under a low temperature of −10 to−80° C. for 1 to 24 hours to obtain frozen crystals; freeze-drying thefrozen crystals to obtain a composite material, the composite materialhaving boiled-off silk woven material as a framework and a layer ofsoluble silk fibroin layer/silk fibroin and gelatin composite layer/silkfibroin and collagen composite layer coated on the framework;

2) placing the composite material obtained in step 1) into a vacuumdryer and performing a vacuum treatment for 20 minutes to 24 hours toobtain a water-insoluble composite high-strength biological scaffoldmaterial, the composite high-strength biological scaffold materialhaving boiled-off silk woven material as a framework and a coating layerprovided on the framework, the material of the coating layer beingselected from the group consisting of: silk protein scaffold material,silk fibroin/gelatin biological scaffold material or silkfibroin/collagen biological scaffold material; and the vacuum dryercontaining water, aqueous methanol or aqueous ethanol at the bottomthereof.

In the above embodiment, in step 1), the aqueous silk fibroin solutionis prepared by: subjecting silk to degumming, dissolution, and dialysisto obtain a silk fibroin solution, the aqueous silk fibroin solutionhaving a mass concentration of 0.1 to 20%; leaving the aqueous silkfibroin solution at 0 to 80° C. for 1 to 48 hours;

In the above embodiment, in step 1), the mixed solution of silk fibroinand gelatin is prepared by: preparing an aqueous gelatin solution byadding medical gelatin into distilled water, heating to obtain anaqueous gelatin solution, and leaving the solution at 0 to 10° C. for 30minutes to 2 hours, the aqueous gelatin solution having a massconcentration of 0.01 to 20%; then mixing an aqueous silk fibroinsolution with the aqueous gelatin solution at a mass ratio of silkfibroin to gelatin of 100: 2 to 20, to obtain the mixed solution of silkfibroin and gelatin;

In the above embodiment, in step 1), the mixed solution of silk fibroinand collagen is prepared by: preparing an aqueous silk fibroin solutionby subjecting silk to degumming, dissolution, and dialysis to obtain asilk fibroin solution, the aqueous silk fibroin solution having a massconcentration of 0.1 to 20%; leaving the aqueous silk fibroin solutionat 0 to 80° C. for 1 to 48 hours; preparing a solution of collagen inacetic acid at a concentration of 0.01% to 2%, and leaving the solutionat 0 to 10° C. for 30 minutes to 2 hours; then mixing the aqueous silkfibroin solution with the solution of collagen in acetic acid at a massratio of silk fibroin to collagen of 100: 2 to 20, to obtain the mixedsolution of silk fibroin and collagen.

In the above embodiment, preferably, the desired shape can be tubular,cylindrical, or conical.

In the above embodiment, preferably, in step 2), the aqueous methanolhas a volume concentration of 1 to 100%; and the aqueous ethanol has avolume concentration of 1 to 100%.

The instant application also claims a method for preparing ahigh-strength anal fistula repair plug, characterized in that, themethod comprises the following steps:

1) preparing a solution containing silk fibroin, the solution containingsilk fibroin being one selected from the group consisting of: an aqueoussilk fibroin solution, a mixed solution of silk fibroin and gelatin, anda mixed solution of silk fibroin and collagen;

2) weaving degummed boiled-off silk into a tubular material to provide aframework and placing the tubular material into a mold for preparinganal fistula repair plug, the weave density of the tubular materialbeing such that the distance between adjacent boiled-off silks isbetween 0.5 mm and 3 mm;

3) injecting the solution containing silk fibroin into the mold havingthe tubular material placed therein, and subjecting it to freezing undera low temperature of −10 to −30° C. for 20 to 24 hours to obtain afrozen body; freeze-drying the frozen body to obtain a soluble compositeanal fistula repair plug, the soluble composite anal fistula repair plughaving the tubular material woven from boiled-off silk as a frameworkand a layer of soluble silk fibroin layer/silk fibroin and gelatincomposite layer/silk fibroin and collagen composite layer coated on theframework;

4) placing the soluble composite anal fistula repair plug into a vacuumdryer and performing a vacuum treatment for 4 hours or more to obtain awater-insoluble composite anal fistula repair plug, the water-insolublecomposite anal fistula repair plug having the tubular material wovenfrom boiled-off silk as a framework and a biological scaffold materialas a coating layer, the biological scaffold material being selected fromthe group consisting of: silk protein biological scaffold material, silkfibroin/gelatin biological scaffold material or silk fibroin/collagenbiological scaffold material, the framework being embedded inside thecoating layer; and the vacuum dryer containing water, aqueous methanolor aqueous ethanol at the bottom thereof.

In the above embodiment, in step 1), the aqueous silk fibroin solutionis prepared by: preparing an aqueous silk fibroin solution having a massconcentration of 0.5% to 5% by a conventional method, and leaving theaqueous silk fibroin solution at 0 to 10° C. for 30 minutes or more;

In the above embodiment, in step 1), the mixed solution of silk fibroinand gelatin is prepared by: preparing an aqueous gelatin solution havinga mass concentration of 0.05% to 1%, and leaving the solution at 0 to10° C. for 30 minutes to 2 hours; mixing an aqueous silk fibroinsolution with the aqueous gelatin solution uniformly such that the massconcentration of silk fibroin is 0.2% to 3% and the mass concentrationof gelatin is 0.02% to 0.2% upon mixing; and leaving the mixture standfor 4 to 10 hours to obtain the mixed solution of silk fibroin andgelatin;

In the above embodiment, in step 1), the mixed solution of silk fibroinand collagen is prepared by: preparing a solution of collagen in aceticacid at a concentration of 0.05% to 1%, and leaving the solution at 0 to10° C. for 30 minutes to 2 hours; mixing an aqueous silk fibroinsolution with the solution of collagen in acetic acid uniformly at 0 to10° C. such that the mass concentration of silk fibroin is 0.2% to 3%and the mass concentration of collagen is 0.02% to 0.2% upon mixing; andleaving the mixture stand for 4 to 10 hours to obtain the mixed solutionof silk fibroin and collagen.

In the above embodiment, preferably, in step 1), the aqueous silkfibroin solution has a mass concentration of 0.1% to 2%; the aqueousgelatin solution or the solution of collagen in acetic acid has a massconcentration of 0.075% to 0.2%; when mixing an aqueous silk fibroinsolution with the aqueous gelatin solution or the solution of collagenin acetic acid uniformly, the aqueous silk fibroin solution is mixedwith the aqueous gelatin solution or the solution of collagen in aceticacid in equal volume.

In the above embodiment, preferably, in step 4), the coating layer has athickness of 100 microns to 5 mm; and the silk fibroin/gelatin orcollagen biological scaffold material has a pore size of 200 to 400microns, and a porosity of 80% or more.

The instant application also claims a composite anal fistula repair plugprepared by the method of the above embodiments, having a tubularmaterial woven from boiled-off silk as a framework and a biologicalscaffold material as a coating layer, the framework being embeddedinside the coating layer, the biological scaffold material beingselected from the group consisting of: silk protein biological scaffoldmaterial, silk fibroin/gelatin biological scaffold material or silkfibroin/collagen biological scaffold material, and the biologicalscaffold material having intercommunicated pores with a pore size of 10to 1000 microns, and a porosity of 80% or more.

In the above embodiment, preferably, the biological scaffold materialhaving intercommunicated pores with a pore size of 150 to 450 microns,and a porosity of 90% or more; wherein the porosity is defined as thevolume percentage of the volume of pores inside a porous solid materialsuch as bricks, rocks, steel, silicon, etc., based on the total volumeof the material, representing how many pores are contained in thematerial.

The anal fistula repair plug has adjustable crystal structure andcomposition, as well as adjustable degradation rate.

The present invention is based on the following principles. Silk fibroinundergoes a slow self-assembly process in an aqueous solution, cangradually be self-assembled into nanospheres or further assembled intonanowires from scattered molecules. Once successfully assembled, silkfibroin can remain stable for a relatively longer time, and can bestable in an aqueous solution. This self-assembly process can beaffected by temperature, hydrophilic and hydrophobic interactions,concentration of the solution, ionic strength, pH, and time, etc. Assuch, the microstructure of silk fibroin can be adjusted by adjustingvarious simple physical factors, thereby controlling the formation ofthe porous structure. It should be noted that, none of the silk fibroinporous materials prepared by any method previously known has mechanicalproperties that can substantially meet the practical requirements ofclinical surgery, and they are urgent for improved mechanical strength,especially tear resistance. Boiled-off silk is a silk fibroin fiberobtained by removing sericin from the surface of silk. It has excellentmechanical strength and tear resistance. In the present invention, theboiled-off silk is woven and incorporated, as a framework, into the silkfibroin-based anal fistula repair plug. The problem of poor mechanicalstrength of the silk fibroin porous materials is thereby effectivelysolved. Finally, the present invention provides a post-processing methodwhich successfully prepares silk fibroin porous scaffolds havingdifferent crystal compositions by treating the silk fibroin scaffolds ina vacuum environment with saturated water, methanol, ethanol and thelike. Thus, the invention achieves controllable degradation of silkfibroin porous scaffolds while effectively reduced or eliminated the useof toxic solvents.

The present invention provides the following advantages over the priorart, due to the use of the above technical solutions.

1) The present invention utilizes tubes woven from biocompatibleboiled-off silk as the framework of the porous anal fistula repair plug,and significantly improves the mechanical strength of the material, tomeet the specific requirements in clinical repair plug applications.

2) The porous scaffold obtained according to the present invention hasintercommunicated pores with a pore size of 10 to 1000 microns, which issuitable for cell adhesion and growth. Thus, the biocompatibility issignificantly improved, which is beneficial for tissue repair and tissueengineering applications. Moreover, the secondary structure of silkfibroin can be effectively regulated by different post treatments,thereby imparting different degradation properties to meet the differentneeds of anal fistula repair. The whole preparation process does notrequire addition of any chemical cross-linking agent or foaming agent,and the good biocompatibility of the silk fibroin is effectivelymaintained.

3) Since it is conducted below room temperature and in an aqueousenvironment, the preparation process is mild and easy to control, anddoes not result in reduced biocompatibility of silk fibroin.

4) In the preparation process, the size of the ice crystals can becontrolled by adjusting parameters such as the freezing temperature orthe concentration of the solution. Thus, the purpose of controlling thestructure of the porous scaffold can be conveniently achieved, so as tomeet different application requirements.

5) In the post treatment process, the crystal structure of the preparedsilk fibroin porous scaffold can be effectively controlled by changingthe processing solution, so as to achieve the purpose of controlling thedegradation rate thereof.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a tubular material woven from boiled-off silkused in Example 1;

FIG. 2 is a photograph of the silk fibroin/gelatin anal fistula repairplug reinforced by boiled-off silk prepared in Example 1;

FIG. 3 is the micro porous structure of the silk fibroin/gelatin analfistula repair plug reinforced by boiled-off silk prepared in Example 1;

FIG. 4 is the IR spectra result of the silk fibroin/gelatin anal fistularepair plug reinforced by boiled-off silk prepared in Example 1.

DETAILED EMBODIMENTS

Next, the present invention is further described by the aid of drawingsand examples.

Example 1

In this example, the silk fibroin porous scaffold is prepared accordingto the following steps:

1) 5 g of cocoons are added to 2 liters of a sodium carbonate solutionhaving a mass concentration of 0.5%, treated at 98 to 100° C. for 40 minto remove the external sericin, and sufficiently washed to obtain puresilk fibroin.

2) The degummed silk fibroin (also referred to as “boiled-off silk”) iswoven into a tubular material having a diameter of 4 mm by a textilemachine to form a framework, the weave density of the material is suchthat the distance between adjacent warp boiled-off silks is about 1 mm,and the distance between adjacent weft boiled-off silks is about 1 mm.The tubular material is placed into a repair plug mold having a diameterof 5 mm.

3) The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromidesolution at about 60° C., to obtain a fibroin mixed solution. Thefibroin mixed solution is added to a cellulose dialysis membrane, anddialysized with deionized water to remove lithium bromide. Thus, a pureaqueous silk fibroin solution is obtained.

4) The mass concentration of the silk fibroin is adjusted to 4%, and theaqueous solution is placed at 4° C. for 1 hour, such that the solutiontemperature is stably maintained at 4° C.

5) A gelatin solution is diluted to 1%.

6) The silk fibroin solution is mixed with the gelatin solution in equalvolume at 4° C., and is allowed to stand for 6 hours.

7) The mixed solution is injected into the mold having boiled-off silktube placed therein, and frozen at −20° C. for 24 hours to obtain a silkfibroin/gelatin frozen body.

8) The frozen silk fibroin/gelatin mixture is placed into a freezedryer, and subjected to a freeze-drying process for 48 hours to obtain asoluble silk fibroin/gelatin porous repair plug reinforced by boiled-offsilk having a thickness of 5 mm.

9) The porous repair plug is placed into a vacuum dryer having waterprovided at the bottom, and subjected to a vacuum treatment for 6 hoursto obtain a water-insoluble silk fibroin/gelatin anal fistula repairplug reinforced by boiled-off silk.

FIGS. 1 and 2 show a boiled-off silk tube having a diameter of 4 mm, anda silk fibroin/gelatin anal fistula repair plug compounded with theboiled-off silk tube, prepared according to the above method. It can beseen, the boiled-off silk is well compounded with the silkfibroin/gelatin porous material.

FIG. 3 is a scanning electron micrograph of a silk fibroin/gelatinporous anal fistula repair plug prepared according to the above method.It can be seen, a good porous structure is formed in the material whichhas a pore diameter of 200-400 microns.

FIG. 4 is the IR spectra of the silk fibroin/gelatin porous anal fistularepair plug reinforced by boiled-off silk prepared according to theabove method. It can be seen, the characteristic absorption peak of thesilk fibroin/gelatin porous repair plug is at 1630 cm⁻¹ and has adistinct shoulder peak at 1650 cm⁻¹, which indicates that both silk Iand silk II crystalline structures exist.

Example 2

5 g of cocoons are added to 2 liters of a sodium carbonate solutionhaving a mass concentration of 0.5%, treated at 98 to 100° C. for 40 minto remove the external sericin, and sufficiently washed to obtain puresilk fibroin.

The degummed silk fibroin (also referred to as “boiled-off silk”) iswoven into a tubular material having a diameter of 3 mm by a textilemachine The tubular material is placed into a conical repair plug moldhaving an upper diameter of 3 mm and a lower diameter of 5 mm.

The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromidesolution at about 60° C., to obtain a fibroin mixed solution. Thefibroin mixed solution is added to a cellulose dialysis membrane, anddialysized with deionized water to remove lithium bromide. Thus, a pureaqueous silk fibroin solution is obtained.

The mass concentration of the silk fibroin is adjusted to 4%, and theaqueous solution is placed at 4° C. for 1 hour, such that the solutiontemperature is stably maintained at 4° C.

A collagen solution in acetic acid is diluted to 0.5%.

The silk fibroin solution is mixed with the collagen solution in equalvolume at 4° C., and is allowed to stand for 6 hours.

The mixed solution is injected into the mold having boiled-off silk tubeplaced therein, and frozen at −20° C. for 24 hours to obtain a silkfibroin/collagen frozen body.

The frozen silk fibroin/collagen mixture is placed into a freeze dryer,and subjected to a freeze-drying process for 48 hours to obtain asoluble silk fibroin/collagen porous repair plug reinforced byboiled-off silk having an upper diameter of 3 mm and a lower diameter of5 mm.

The porous repair plug is placed into a vacuum dryer having waterprovided at the bottom, and subjected to a vacuum treatment for 6 hoursto obtain a water-insoluble silk fibroin/collagen anal fistula repairplug reinforced by boiled-off silk.

Example 3

5 g of cocoons are added to 2 liters of a sodium carbonate solutionhaving a mass concentration of 0.5%, treated at 98 to 100° C. for 40 minto remove the external sericin, and sufficiently washed to obtain puresilk fibroin.

The degummed silk fibroin (also referred to as “boiled-off silk”) iswoven into a tubular material having a diameter of 4 mm by a textilemachine The tubular material is placed into a conical repair plug mold.

The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromidesolution at about 60° C., to obtain a fibroin mixed solution. Thefibroin mixed solution is added to a cellulose dialysis membrane, anddialysized with deionized water to remove lithium bromide. Thus, a pureaqueous silk fibroin solution is obtained.

The mass concentration of the silk fibroin is adjusted to 4%, and theaqueous solution is placed at 4° C. for 1 hour, such that the solutiontemperature is stably maintained at 4° C.

A collagen solution in acetic acid is diluted to 0.5%.

The silk fibroin solution is mixed with the collagen solution in equalvolume at 4° C., and is allowed to stand for 6 hours.

The mixed solution is injected into the mold having boiled-off silk tubeplaced therein, and frozen at −20° C. for 24 hours to obtain a silkfibroin/collagen frozen body.

The frozen silk fibroin/collagen mixture is placed into a freeze dryer,and subjected to a freeze-drying process for 48 hours to obtain asoluble silk fibroin/collagen porous repair plug reinforced byboiled-off silk.

The porous repair plug is placed into a vacuum dryer having an 80%ethanol solution provided at the bottom, and subjected to a vacuumtreatment for 6 hours to obtain a water-insoluble silk fibroin/collagenporous anal fistula repair plug reinforced by boiled-off silk. Thisporous scaffold has a pore diameter of 200 microns or more, and aporosity of 90% or more. The main crystal structure is silk II.

Example 4

5 g of cocoons are added to 2 liters of a sodium carbonate solutionhaving a mass concentration of 0.5%, treated at 98 to 100° C. for 40 minto remove the external sericin, and sufficiently washed to obtain puresilk fibroin.

The degummed silk fibroin (also referred to as “boiled-off silk”) iswoven into a tubular material having a diameter of 4 mm by a textilemachine The tubular material is placed into a conical repair plug mold.

The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromidesolution at about 60° C., to obtain a fibroin mixed solution. Thefibroin mixed solution is added to a cellulose dialysis membrane, anddialysized with deionized water to remove lithium bromide. Thus, a pureaqueous silk fibroin solution is obtained.

The mass concentration of the silk fibroin is adjusted to 4%, and theaqueous solution is placed at 4° C. for 1 hour, such that the solutiontemperature is stably maintained at 4° C.

An aqueous gelatin solution is diluted to 0.5%.

The silk fibroin solution is mixed with the gelatin solution in equalvolume at 4° C., and is allowed to stand for 6 hours.

The mixed solution is injected into the mold having boiled-off silk tubeplaced therein, and frozen at −20° C. for 24 hours to obtain a silkfibroin/gelatin frozen body.

The frozen silk fibroin/gelatin mixture is placed into a freeze dryer,and subjected to a freeze-drying process for 48 hours to obtain asoluble silk fibroin/gelatin porous repair plug reinforced by boiled-offsilk.

The porous repair plug is placed into a vacuum dryer having an 80%methanol solution provided at the bottom, and subjected to a vacuumtreatment for 6 hours to obtain a water-insoluble silk fibroin/collagenporous anal fistula repair plug reinforced by boiled-off silk. Thisporous scaffold has a pore diameter of 200 microns or more, and aporosity of 90% or more. The main crystal structure is silk II.

Example 5

5 g of cocoons are added to 2 liters of a sodium carbonate solutionhaving a mass concentration of 0.5%, treated at 98 to 100° C. for 40 minto remove the external sericin, and sufficiently washed to obtain puresilk fibroin.

The degummed silk fibroin (also referred to as “boiled-off silk”) iswoven into a tubular material having a diameter of 3 mm by a textilemachine The tubular material is placed into a repair plug mold having aninner diameter of 2.9 mm and an outer diameter of 3.2 mm.

The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromidesolution at about 60° C., to obtain a fibroin mixed solution. Thefibroin mixed solution is added to a cellulose dialysis membrane, anddialysized with deionized water to remove lithium bromide. Thus, a pureaqueous silk fibroin solution is obtained.

The mass concentration of the silk fibroin is adjusted to 4%, and theaqueous solution is placed at 4° C. for 1 hour, such that the solutiontemperature is stably maintained at 4° C.

A collagen solution in acetic acid is diluted to 0.5%.

The silk fibroin solution is mixed with the collagen solution in equalvolume at 4° C., and is allowed to stand for 6 hours.

The mixed solution is injected into the mold having boiled-off silk tubeplaced therein, and frozen at −20° C. for 24 hours to obtain a silkfibroin/collagen frozen body having a thickness of 300 microns.

The frozen silk fibroin/collagen mixture is placed into a freeze dryer,and subjected to a freeze-drying process for 48 hours to obtain asoluble silk fibroin/collagen porous repair plug reinforced byboiled-off silk.

The porous repair plug is placed into a vacuum dryer having an 80%ethanol solution provided at the bottom, and subjected to a vacuumtreatment for 6 hours to obtain a water-insoluble silk fibroin/collagenporous anal fistula repair plug reinforced by boiled-off silk. Thisporous scaffold has a pore diameter of 200 microns or more, and aporosity of 90% or more. The main crystal structure is silk II.

Example 6

5 g of cocoons are added to 2 liters of a sodium carbonate solutionhaving a mass concentration of 0.5%, treated at 98 to 100° C. for 40 minto remove the external sericin, and sufficiently washed to obtain puresilk fibroin.

The degummed silk fibroin (also referred to as “boiled-off silk”) iswoven into a sheet material having an area of 4×4 cm. The sheet materialis placed into a mold having a height of 4 cm.

The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromidesolution at about 60° C., to obtain a fibroin mixed solution. Thefibroin mixed solution is added to a cellulose dialysis membrane, anddialysized with deionized water to remove lithium bromide. Thus, a pureaqueous silk fibroin solution is obtained.

The mass concentration of the silk fibroin is adjusted to 4%, and theaqueous solution is placed at 4° C. for 1 hour, such that the solutiontemperature is stably maintained at 4° C. The silk fibroin solution isslowly concentrated to 30%, placed at 4° C. for 3 days, and re-dilutedto 4%.

The diluted silk fibroin solution is injected into the mold havingboiled-off silk sheet placed therein, and frozen at −20° C. for 24 hoursto obtain a silk fibroin frozen body having a height of 4 cm and an areaof 4×4 cm.

The frozen body is placed into a freeze dryer, and subjected to afreeze-drying process for 48 hours to obtain a soluble silk fibroin bulkmaterial reinforced by boiled-off silk.

The bulk material is placed into a vacuum dryer having an 80% methanolsolution provided at the bottom, and subjected to a vacuum treatment for6 hours to obtain a water-insoluble silk fibroin repair sheet material.This porous repair plug has a pore diameter of 200 microns or more, anda porosity of 90% or more. The main crystal structure is silk II.

1. A high-strength biological scaffold material, characterized in that,said high-strength biological scaffold material is comprised of acoating layer and a framework, said framework is embedded in the coatinglayer; said framework is a boiled-off silk woven material, the weavedensity can be determined by needs; the coating layer has a thickness of100 microns to 5 cm, and the material of the coating layer is selectedfrom the group consisting of: silk protein scaffold material, silkfibroin/gelatin biological scaffold material or silk fibroin/collagenbiological scaffold material.
 2. The high-strength biological scaffoldmaterial according to claim 1, characterized in that, the weave densityof the boiled-off silk woven material is such that the distance betweenadjacent boiled-off silks is between 0.5 mm and 3 mm.
 3. Thehigh-strength biological scaffold material according to claim 1,characterized in that, the coating layer has a thickness of 100 micronsto 5 mm; and the high-strength biological scaffold material has a poresize of 200 to 400 microns, and a porosity of 80% or more.
 4. A methodfor preparing a high-strength biological scaffold material,characterized in that, the method comprises the following steps:boiled-off silk obtained by degumming silk is woven into a wovenmaterial by a textile machine to form a framework according to thedesired shape, and then the framework is placed into a mold; a solutioncontaining silk fibroin is injected into the mold having the frameworkplaced therein, and subjected to freezing and vacuum treatment; therebya coating layer having a thickness of 100 microns to 5 cm is formed onthe surface of the framework, and the high-strength biological scaffoldmaterial is prepared, wherein said solution containing silk fibroin isone selected from the group consisting of: an aqueous silk fibroinsolution, a mixed solution of silk fibroin and gelatin, and a mixedsolution of silk fibroin and collagen; the weave density of the wovenmaterial is such that the distance between adjacent boiled-off silks isbetween 0.5 mm and 3 mm, and the material of the coating layer isselected from the group consisting of: silk protein scaffold material,silk fibroin/gelatin biological scaffold material or silkfibroin/collagen biological scaffold material.
 5. The method forpreparing a high-strength biological scaffold material according toclaim 4, characterized in that, the step of coating the surface of theframework with a coating layer comprises the specific steps of: 1)preparing a solution containing silk fibroin, the solution containingsilk fibroin being one selected from the group consisting of: an aqueoussilk fibroin solution, a mixed solution of silk fibroin and gelatin, anda mixed solution of silk fibroin and collagen; injecting the solutioncontaining silk fibroin into the mold having the framework placedtherein, and subjecting it to freezing under a low temperature of −10 to−80° C. for 1 to 24 hours to obtain frozen crystals; freeze-drying thefrozen crystals to obtain a composite material, the composite materialhaving boiled-off silk woven material as a framework and a layer ofsoluble silk fibroin layer/silk fibroin and gelatin composite layer/silkfibroin and collagen composite layer coated on the framework; 2) placingthe composite material obtained in step 1) into a vacuum dryer andperforming a vacuum treatment for 20 minutes to 24 hours to obtain awater-insoluble composite high-strength biological scaffold material,the composite high-strength biological scaffold material havingboiled-off silk woven material as a framework and a coating layerprovided on the framework, the material of the coating layer beingselected from the group consisting of: silk protein scaffold material,silk fibroin/gelatin biological scaffold material or silkfibroin/collagen biological scaffold material; and the vacuum dryercontaining water, aqueous methanol or aqueous ethanol at the bottomthereof; wherein in step 1), the aqueous silk fibroin solution isprepared by: subjecting silk to degumming, dissolution, and dialysis toobtain a silk fibroin solution, the aqueous silk fibroin solution havinga mass concentration of 0.1 to 20%; leaving the aqueous silk fibroinsolution at 0 to 80° C. for 1 to 48 hours; the mixed solution of silkfibroin and gelatin is prepared by: preparing an aqueous gelatinsolution by adding medical gelatin into distilled water, heating toobtain an aqueous gelatin solution, and leaving the solution at 0 to 10°C. for 30 minutes to 2 hours, the aqueous gelatin solution having a massconcentration of 0.01 to 20%; then mixing an aqueous silk fibroinsolution with the aqueous gelatin solution at a mass ratio of silkfibroin to gelatin of 100: 2 to 20, to obtain the mixed solution of silkfibroin and gelatin; the mixed solution of silk fibroin and collagen isprepared by: preparing an aqueous silk fibroin solution by subjectingsilk to degumming, dissolution, and dialysis to obtain a silk fibroinsolution, the aqueous silk fibroin solution having a mass concentrationof 0.1 to 20%; leaving the aqueous silk fibroin solution at 0 to 80° C.for 1 to 48 hours; preparing a solution of collagen in acetic acid at aconcentration of 0.01% to 2%, and leaving the solution at 0 to 10° C.for 30 minutes to 2 hours; then mixing the aqueous silk fibroin solutionwith the solution of collagen in acetic acid at a mass ratio of silkfibroin to collagen of 100: 2 to 20, to obtain the mixed solution ofsilk fibroin and collagen.
 6. A method for preparing a high-strengthanal fistula repair plug, characterized in that, the method comprisesthe following steps: 1) preparing a solution containing silk fibroin,the solution containing silk fibroin being one selected from the groupconsisting of: an aqueous silk fibroin solution, a mixed solution ofsilk fibroin and gelatin, and a mixed solution of silk fibroin andcollagen; 2) weaving degummed boiled-off silk into a tubular material toprovide a framework and placing the tubular material into a mold forpreparing anal fistula repair plug, the weave density of the tubularmaterial being such that the distance between adjacent boiled-off silksis between 0.5 mm and 3 mm; 3) injecting the solution containing silkfibroin into the mold having the tubular material placed therein, andsubjecting it to freezing under a low temperature of −10 to −30° C. for20 to 24 hours to obtain a frozen body; freeze-drying the frozen body toobtain a soluble composite anal fistula repair plug, the solublecomposite anal fistula repair plug having the tubular material wovenfrom boiled-off silk as a framework and a layer of soluble silk fibroinlayer/silk fibroin and gelatin composite layer/silk fibroin and collagencomposite layer coated on the framework; 4) placing the solublecomposite anal fistula repair plug into a vacuum dryer and performing avacuum treatment for 4 hours or more to obtain a water-insolublecomposite anal fistula repair plug, the water-insoluble composite analfistula repair plug having the tubular material woven from boiled-offsilk as a framework and a biological scaffold material as a coatinglayer, the biological scaffold material being selected from the groupconsisting of: silk protein biological scaffold material, silkfibroin/gelatin biological scaffold material or silk fibroin/collagenbiological scaffold material, the framework being embedded inside thecoating layer; and the vacuum dryer containing water, aqueous methanolor aqueous ethanol at the bottom thereof; wherein in step 1), theaqueous silk fibroin solution is prepared by: preparing an aqueous silkfibroin solution having a mass concentration of 0.5% to 5% by aconventional method, and leaving the aqueous silk fibroin solution at 0to 10° C. for 30 minutes or more; the mixed solution of silk fibroin andgelatin is prepared by: preparing an aqueous gelatin solution having amass concentration of 0.05% to 1%, and leaving the solution at 0 to 10°C. for 30 minutes to 2 hours; mixing an aqueous silk fibroin solutionwith the aqueous gelatin solution uniformly such that the massconcentration of silk fibroin is 0.2% to 3% and the mass concentrationof gelatin is 0.02% to 0.2% upon mixing; and leaving the mixture standfor 4 to 10 hours to obtain the mixed solution of silk fibroin andgelatin; the mixed solution of silk fibroin and collagen is prepared by:preparing a solution of collagen in acetic acid at a concentration of0.05% to 1%, and leaving the solution at 0 to 10° C. for 30 minutes to 2hours; mixing an aqueous silk fibroin solution with the solution ofcollagen in acetic acid uniformly at 0 to 10° C. such that the massconcentration of silk fibroin is 0.2% to 3% and the mass concentrationof collagen is 0.02% to 0.2% upon mixing; and leaving the mixture standfor 4 to 10 hours to obtain the mixed solution of silk fibroin andcollagen.
 7. The method for preparing a high-strength anal fistularepair plug according to claim 6, characterized in that, in step 4), thecoating layer has a thickness of 100 microns to 5 mm; and the biologicalscaffold material has a pore size of 200 to 400 microns, and a porosityof 80% or more.
 8. A composite anal fistula repair plug prepared by themethod of claim 6, having a tubular material woven from boiled-off silkas a framework and a biological scaffold material as a coating layer,the framework being embedded inside the coating layer, the biologicalscaffold material being selected from the group consisting of: silkprotein biological scaffold material, silk fibroin/gelatin biologicalscaffold material or silk fibroin/collagen biological scaffold material,and the biological scaffold material having intercommunicated pores witha pore size of 10 to 1000 microns, and a porosity of 80% or more.
 9. Thecomposite anal fistula repair plug according to claim 8, wherein thecoating layer has a thickness of 100 microns to 5 mm; and the biologicalscaffold material has a pore size of 200 to 400 microns, and a porosityof 80% or more.